The overall goal of this proposal is to develop and implement a systematic approach that will result in the identification and characterization of lysosomal proteins and to understand their role in human disease. This approach relies on the finding that most lysosomal proteins contain a common post-translational modification, the mannose 6-phosphate (M6P) recognition marker, which distinguishes them from other types of proteins. There are three specific aims. Specific Aim 1 is to identify the spectrum of M6P glycoproteins encoded by the human genome using both two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry based approaches. This will identify a number of previously undiscovered candidate lysosomal proteins and will create a proteomics resource to investigate their role in human disease. Specific Aim 2 is to begin to characterize the function of previously unidentified M6P glycoproteins, This will entail determining their subcellular distribution to verify their lysosomal location as well as generating reagents for structural and functional studies, Specific Aim 3 is to investigate the molecular bases for lysosomal storage diseases of unknown etiology. Two approaches will be used: 1) The "disease-to-protein" approach will entail global comparisons of M6P glycoproteins in normal and disease specimens using the resources created in Specific Aim 1. Extensions of this approach may be applicable towards development of clinical screening methods for lysosomal storage diseases. 2) The protein-to-disease approach will create a database of the properties of newly characterized M6P glycoproteins and use this information to identify candidates for associated diseases. Potential disease genes identified using either approach will be scrutinized using molecular genetic analysis. Given that this approach has resulted in the identification of several previously uncharacterized lysosornal proteins and the molecular basis for three hereditary diseases, it is anticipated that extensions of this discovery-based research will result in additional important findings.